Authors: Abu Bakar Siddique, Hassan Ebrahim, Mohamed Mohyeldin, Seetharama D. Jois and Khalid A. El Sayed
Dysregulation of the receptor tyrosine kinases (RTKs) HER2 and c-Met correlate well with poor breast cancer (BC) prognosis and invasive aggressive profile. c-Met amplification proved to be the HER2-dependent tumors' inevitable escape mechanism from the anticancer effects of targeted therapies including trastuzumab, cetuximab, and small-molecule RTK inhibitors like lapatinib, gefitinib, and erlotinib. Dual HER2-Met inhibition is highly expected to be effective and less likely to develop resistance. This study reports the novel dual HER2-Met inhibitory molecular mechanism for the extra virgin olive oil-derived phenolic secoiridoid (-)-oleocanthal (OC). OC showed typical type-I binding mode at c-Met ATP kinase domain. OC aldehydes, ester, and phenol groups showed critical interactions with the critical activation loop ASP1222/TYR1230 and the hinge region PRO1158/MET1160. In addition, OC overlapped two out of the three lapatinib critical interactions at the HER2 kinase domain, the hinge region MET801 and PHE864; besides, OC uniquely targeted the hinge region's THR862 and SER783, justifying unique binding mode. OC showed low-µM level inhibitory activities against both c-Met and HER2 kinases in cell-free Z-LYTE assays. In vitro, OC induced dose- and time-dependent inhibition of the proliferation and migrations of several BC cells including BT-474, SK-BR-3, MDA-MB-231, MCF-7, MDA-MB-468 at low μM IC50 dose range. This effect was associated with selective blockade of HER2 and Met receptors activation in response to their natural ligands HGF and EGF, respectively, confirmed by Western blotting, flow cytometry and immunocytochemistry studies. OC potently induced autophagy via the upregulation of LCA/B, Atg-3, Atg-7, Atg-16L within 6-12 h of SKBR-3 cells treatment. OC had no effect on the viability of the nontumorigenic human MCF-12A mammary epithelial and neuronal Schwann RSC 96 cells. In vivo, 5-10 mg/kg oral or ip dose range of OC potently inhibited 65-90% of the growth of the HER2-dependent BT-474 and the c-Met-dependent MDA-MB-231 BC cells in female athymic nude mice xenograft models. Orally, 10 mg/kg dose of OC prevented over 90% of local and regional recurrences of both tumor phenotypes after primary tumor surgical excision in orthotopic xenograft mice models. This was further confirmed by significant reductions of Ki-67, CD31, pHER2 and p-c-Met levels in treated animal tumors by IHC studies. OC is a novel dual c-Met-HER2 inhibitory dietary supplement lead with excellent potential for translational use to prevent and control breast malignancies with aberrant c-Met or HER2 activities.